PGF{HD 2{301 {B {0 and PGF{HD 2{331 {B , 15-acetate, methyl esters

ABSTRACT

PGF2 , 15-acetate, methyl ester, and PGF2 , 15-acetate, methyl ester, novel compounds useful for the same pharmacological purposes as PGF2 and PGF2 , respectively.

United States Patent Pike et al.

[ Dec. 16, 1975 PGF a AND PGF B IS-ACETATE, METHYL ESTERS Inventors:John E. Pike; William P. Schneider,

both of Kalamazoo, Mich.

Assignee: The Upjohn Company, Kalamazoo,

Mich.

Filed: Oct. 25, 1973 Appl. No.: 404,962

Related US. Application Data Division of Scr. No. 159,478, July 2, 1971,Pat. No. 3,772,350, which is a continuation-in-part of Ser. No. 71390.Sept. 11. 1970, abandoned.

US. Cl... 260/488 R; 260/348 A; 260/348.5 L; 260/448.8 R; 260/468 D;260/491; 260/499; 260/514 D; 424/305; 424/311 lnt. Cl. ..'C07C 177/00[58] Field of Search 260/488 R, 514 D, 468 D Primary ExaminerVivianGarner Attorney, Agent, or Firm-Morris L. Nielsen; Earl C. Spaeth ABS'IRACT PGF a IS-acetate, methyl ester, and PGF B 15-acetate, methyl ester,novel compounds useful for the same pharmacological purposes as PGF aand PGF B respectively.

3 Claims, No Drawings PGF AND PGFzB ,IS-ACETATE, METHYL ESTERS CROSSREFERENCE TO RELATED APPLICATIONS This application is a division of ourco-pending application Ser. No. l59,478, filed July 2, 1971, now US.Pat. No. 3,772,350 which was a continuation-in-part of our copendingapplication Ser. No. 71,390, filed Sept. ll, 1970 and now abandoned.

DESCRIPTION OF THE INVENTION This invention relates to novelcompositions of matter, to novel methods for preparing them, and tonovel intermediates used in those methods. This invention also relatesto novel methods for preparing known compounds, and to novelintermediates used in those methods.

In particular, the several aspects of this invention relate toderivatives of prostanoic acid which has the following structure andnumbering:

COOH 1 Some of the derivatives of prostanoic acid are known asprostaglandins. One of those, prostaglandin E (PGE has the followingformula:

Another,v prostaglandin F (PGF the for-v mula:

1 COOH.

Still another, prostaglandin F '3 (PGF )f has the formula:

shown by attachment of said side-chain hydroxy to C15 with a dotted lineand hydrogen with a heavy solid line. The alternative configuration forthe side-chain hydroxy at Cv-l5 is known as R or epi (beta), and isshown when necessary by attachment of said side-chain hydroxy to C-1.5with a heavy solid line and hydrogen with a dotted line, thus 7 H OH.

The prostaglandin corresponding to PGE (Formula II) but with the R orepi configuration at C-l5 will be designated l5BPGE See Nature, 212, 38(l966jzfor discussion of the stereochemistry of the prostaglandins.These conventions regarding formulas, namesyand symbols for derivativesof prostanoic acid apply to the formulas, names, and symbols givenhereinafterin the specification and claims. When reference is madehereinafter to the compounds of Formulas II to IV, by the symbols PGBPGF a or PGF p or to the methyl esters of any of those, l5(S)configuration will be intended and by established custom, S or alphawill not be mentioned in the name or symbol. For all of the othercompounds recited hereinafter, the configuration at C-l5 will beidentified in the name as 153 whenever the l5(R) configuration isintended. V

Molecules of the known prostaglandins each have several centers ofasymmetry, and can exist in racemic (optically inactive) form and ineither of the two enantiomeric (optically active) forms, he, thedextrorotatory and levorotatory forms. As drawn, Formulas II to IV eachrepresent the particular optically active form of theprostaglandin'which is obtaine'd'froin certain mammalian tissues, forexample, sheep vesicular glands, swine lung, or human seminal plasma,orby carbonyl and/or double bond reduction of a prostaglandin soobtained. See, for example, B'ergstrom et al., Pharmacol. Rev.i20, -l(1968) and references cited therein. I

The several aspects of this invention relate to novel methods forpreparing PGE PGF: 0: and PGF B their'acetates and methyl esters, andthe l58-epimers of those compounds, to novel intermediates used in thosemethods, to novel methods used to make those intermediates, and tocertain novel and pharmacologically useful analogs of PGE PGF a and PGFB The novel and pharmacologically useful PGE PGE; and PGF B analogs ofthis invention have the formulas:

WCOOR In Formulas V, VI, and VII, R is hydrogen, alkyl of l to 8 carbonatoms, inclusive, cycloalkyl of 3 to carbon atoms, inclusive, aralkyl of7 to 12 carbon atoms, inclusive phenyl, or phenyl substituted with l to3 chloro or alkyl of l to 4 carbon atoms, inclusive. Also encompassed byFormulas V, VI, and VII are pharmacologically acceptable salts when R,is hydrogen. In Formulas V and VII, Y is v O OH In Formula V, B is InFormula VI, indicates attachment to the ring in alpha or betaconfiguration.

It will be observed that each of the novel compounds of Formulas V andVI has a hydroxy group attached to the ll-position in betaconfiguration. In PGE P6P, and PGF and in the compounds of Formula VII,the hydroxy at C-ll is attached in alpha configuration.

With regard to Formulas V, VI, and VII, examples of alkyl of one to 8carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, and

isomeric fonns thereof. Examples of cycloalkyl of 3 to 10 carbon atoms,inclusive, which includes alkyl-substituted cycloalkyl, are cyclopropyl,Z-methylcyclopropyl, 2,2-dimethylcyclopropyl, 2,3-diethylcyclopropyl,2-butylcyclopropyl, cyclobutyl, 2-methylcyclobutyl, 3-propylcyclobutyl,2,3,4-triethylcyclobutyl, cyclopentyl, 2,2-dimethylcyclopentyl,3-pentylcyclopentyl, 3- terbbutylcyclopentyl, cyclohexyl,4-tert-butylcyclohexyl, 3-isopropylcyclohexyl, 2,2-dimethylcyclohex'yl,cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Examples of aralkylof 7 to 12 carbon atoms, inclusive, are benzyl, phenethyl,l-phenylethyl, 2- phenylpropyl, 4-phenylbutyl, 3-phenylbutyl, 2-(1-naphthylethyl), and l-( 2-naphthylmethyl). Examples of phenylsubstituted by one to 3 chloro or alkyl of l to VII 4 carbon atoms,inclusive, are p-chlorophenyl, mchlorophenyl, o-chlorophenyl,2,4-dichlorophenyl,2,4,6-trichlorophenyl, p-tolyl, m-tolyLo-tolyl,p-ethylphenyl, p-tert-butylphenyl, 2,5-dimethylphenyl,4-chloro-2-methylphenyl, and 2,4-dichloro-3-methylphenyl.

The known prostanoic acid derivatives, PGE PGF a and PGF B and theiresters and pharmacologically acceptable salts are extremely potent incausing various biological responses. For that reason, these compoundsare useful for pharmacological purposes. See, for example, Bergstrom etal., cited above, and references cited therein. A few of thosebiological responses are systemic arterial blood pressure lowering inthe case of the PGE and PGE compounds as measured, for example, inanesthetized (pentobarbital sodium) pentolinium-treated rats withindwelling aortic and right heart cannulas; pressor activity, similarlymeasured, for the PGF compounds; stimulation of smooth muscle as shown,for example, by tests on strips of guinea pig ileum, rabbit duodenum, orgerbil colon; potentiation of other smooth muscle stimulants;antilipolytic activity as shown by antagonism of epinephrine-inducedmobilization of free fattyacids or inhibition of the spontaneous releaseof glycerol from isolated rat fat pads; inhibition of gastricsecretionin the case of the PGE compounds as shown in dogs with secretionstimulated by food or histamine infusion; activity on the centralnervous system; controlling spasm and facilitating breathing inasthmatic condi tions; decrease of blood platelet adhesiveness as shownby platelet-to-glass adhesiveness, and inhibition of blood plateletaggregation and thrombus formation induced by various physical stimuli,e. g., arterial injury, and various biochemical stimuli, e.g., ADP, ATP,serotonin, thrombin, and collagen; and in the case of the PGE compounds,stimulation of epidermal proliferation and keratinization as shown whenapplied in culture to embryonic chick and rat skin segments.

Because of these biological responses, these known prostaglandins areuseful to study, prevent, control, or alleviate a wide variety ofdiseases and undesirable physiological conditions in birds and mammals,including humans, useful domestic animals, pets, and zoologicalspecimens, and in laboratory animals, for example, mice, rats, rabbits,and monkeys.

For example, these compounds, and especially the PGE compounds, areuseful in mammals, including man, as nasal decongestants. For thispurpose, the compounds are used in a dose range of about ug. to about 10mg. per ml. of a pharmacologically suitable liquid vehicle or as anaerosol spray, both for topical application.

The PCB and PGF compounds are useful in the treatment of asthma. Forexample, these compounds are useful as bronchodilators or as inhibitorsof mediators, such as SRS-A and Histamine which are released from cellsactivated by an antigen-antibody complex. Thus, these compounds controlspasm and facilitate breathing in conditions such as bronchial asthma,bronchitis, bronchiectasis, pneumonia and emphysema. For thse purposes,these compounds are administered in a variety of dosage forms, e.g.,orally in the form of tablets, capsules, or liquids; rectally in theform of tablets, capsules, or liquids; rectally in the form ofsuppositories; parenterally, subcutaneously, or intramuscularly, withintravenous administration being preferred in emergency situations; byinhalation in the form of aerosols or solutions for nebulizers; or byinsufilation in the form of powder. Doses in the range of about 0.01 to5 mg. per kg. of body weight are used 1 to 4 times a day, the exact dosedepending on the age, weight, and condition of the patient and on thefrequency and route of administration. For the above use theseprostaglandins can be combined advantageously with other antiasthmaticagents, such as sympathomimetics (isoproterenol, phenylephrine,ephedrine, etc); xanthine derivatives (theophylline and aminophyllin);and corticosteroids (ACTH and predinisolone). Regarding use of thesecompounds see South African Patent No. 681,055.

The PGE compounds are useful in mammals, including man and certainuseful animals, e.g., dogs and pigs, to reduce and control excessivegastric secretion, thereby reducing or avoiding gastrointestinal ulcerformation, and accelerating the healing of such ulcers already presentin the gastrointestinal tract. For this purpose, the compounds areinjected or infused intravenously, subcutaneously, or intramuscularly inan infusion dose range about 0.1 pg. to about 500 pg. per kg. of bodyweight per minute, or in a total daily dose by injection or infusion inthe range about 0.1 to about mg. per kg. .of body weight per day, theexact dose depending on the age, weight, and condition of the patient oranimal, and on the frequency and route and administration.

The PGE PGF a and PGF [3 compounds are useful whenever it is desired toinhibit platelet aggregation, to reduce the adhesive character ofplatelets, and to remove or prevent the formation of thrombi in mammals,including man, rabbits, and rats. For example, these compounds areuseful in the treatment and prevention of myocardial infarcts, to treatand prevent post-operative thrombosis, to promote patency of vasculargrafts following surgery, and to treat conditions such asatherosclerosis, arteriosclerosis, blood clotting defects due tolipemia, and other clinical conditions in which the underlying etiologyis associates with lipid imbalance or hyperlipidemia. For thesepurposes, these compounds are administered systemically, e.g.,intravenously, subcutaneously, intramuscularly, and in the form ofsterile implants for prolonged action. For rapid response, especially inemergency situations, the intravenous route of administration ispreferred. Doses in the range about 0.005 to about 20 mg. per kg. ofbody weight per day are used, the exact dose depending on the age,weight, and condition of the patient or animal, and on the frequency androute of administration.

The PGE PGF a and PGF compounds are especially useful as additives toblood, blood products, blood substitutes, and other fluids which areused in artificial extracorporeal circulation and perfusion of isolatedbody portions, e.g., limbs and organs, whether attached to the originalbody, detached and being preserved or prepared for transplant, orattached to a new body. Duringthese circulations and perfusions,aggregated platelets tend to block the blood vessels and portions of thecirculation apparatus. This blocking is avoided by the presence of thesecompounds. For this purpose, the compound is added gradually or insingle or multiple portions to the circulating blood, to the blood ofthe donor animal, to the perfused body portion, attached" or detached,to the recipient, or to two or all of those at a total steady state doseof about 0.001 to 10 mg. per liter of circulating fluid. It isespecially useful to use these compounds in laboratory animlas, e.g.,cats, dogs, rabbits, monkeys, and rats, for thses purposes in order todevelop new methods and techniques for organ and limb transplants.

The PGE compounds are extremely potent in causing stimulation of smoothmuscle, and are also highly active in potentiating other known smoothmuscle stimulators, for example, oxytocic agents, e.g., oxytocin, andthe various ergot alkaloids including derivatives and analogs thereof.Therefore PGE for example, is useful in place of or in combination withless than usual amounts of these known smooth muscle stimulators, forexample, to relieve the symptoms of paralytic ileus, or to control orprevent atonic uterine bleeding after abortion or delivery, to aid inexpulsion of the placenta, and during the puerperium. For the latterpurpose, the PGE compound is administered by intravenous infusionimmediately after abortion or delivery at a dose in the range about 0.01to about 50 pg. per kg. of body weight per minute until the desiredeffect is obtained. Subsequent doses are given by intravenous,subcutaneous, or intramuscular injection or infusion during puerperiumin the range 0.01 to 2 mg. per kg. of body weight per day, the exactdose depending on the age, weight, and condition of the patient oranimal.

The PGE and PGF 5 compounds are useful as hypotensive agents to reduceblood pressure in mammals, including man. For this purpose, thecompounds are administered by intravenous infusion at the rate about0.01 to about 50 pg. per kg. of body weight per minute, or in single ormultiple "doses of about 25 to 500 pg. per kg. of body weight total perday.

The PGE PGA and PGF B compounds also increase the flow of blood in themammalian kidney, thereby increasing volume and electrolyte content ofthe urine. Therefore, these compounds are useful in managing cases ofrenal disfunction, especially those involving blockage of the renalvascular bed. Illustratively, the compounds are useful to alleviate andcorrect cases of edema resulting, for example, from massive surfaceburns, and in the management of shock. For these purposes, the compoundsare preferably first administered by intravenous injection at a dose inthe range 10 to 1000 pg. per kg. of body weight or by intravenousinfusion at a dose in the range 0.1 to pg. per kg. of body weight perminute until the desired effect is obtained. Subsequent doses are givenby intravenous, intramuscular, or subcutaneous injection or infusion inthe range 0.05 to 2 mg. per kg. of body weight per day.

The PGE PGF a and PGF 3 compounds are useful in place of oxytocin toinduce labor in pregnant female animals, including man, cows, sheep, andpigs, at or near term, or in pregnant animals with intrauterine death ofthe fetus from about 20 weeks to term. For this purpose, the compound isinfused intravenously at a dose 0.01 to 50 pg. per kg. of body weightper minute until or near the termination of the second stage of labor,i.e., expulsion of the fetus. These compounds are especially useful whenthe female is one or more weeks post-mature and natural labor has notstarted, or 12 to 60 hours after the membranes have ruptured and naturallabor has not yet started.

The PGF a PGF and PGE compounds are useful for controlling thereproductive cycle in ovulating female mammals, including humans andanimals such as monkeys, rats, rabbits, dogs, cattle, and the like. Forthat purpose PGE or PGF a for example, is administered systemically,e.g., intravenously, subcutaneously, and intravaginally, at a dose levelin the range 0.001 mg. to about 20 mg. per kg. of body weight of thefemale mammal, advantageously during a span of time startingapproximately at the time of ovulation and ending approximately at thenext expected time of menses or just prior to that time. Additionally,expulsion of an embryo or fetus (abortion) is accomplished by similaradministration of the compound during the first third of the normalmammalian gestation period.

As mentioned above, the PGE compounds are potent antagonists ofepinephrine-induced mobilization of free fatty acids. For this reason,this compound is useful in experimental medicine for both in vitro andin vivo studies in mammals, including man, rabbits, and rats, intendedto lead to the understanding, prevention, symptom alleviation, and cureof diseases involving abnormal lipid mobilization and high free fattyacid levels, e.g., diabetes mellitus, vascular diseases, andhyperthyroidism.

The novel Formula-V, -VI, and -VII PGE PGF a and PGF analogs of thisinvention each cause the biological responses described above for PGEP6P and PGF respectively, and each of these novel compounds isaccordingly useful for the abovedescribed corresponding purposes, and isused for those purposes in the same manner as described above.

PGE PGF a and PGF B and their esters and pharmacologically acceptablesalts are all potent in causing multiple biological responses even atlow doses. For example, PGE is extremely potent in causingvasodepression and smooth muscle stimulation, and is also potent as anantilipolytic agent. Moreover, for many applications, these knownprostaglandins have an inconveniently short duration of biologicalactivity. In striking contrast, the novel analogs of Formulas V, VI, andVII are substantially more specific with regard to potency in causingprostaglandin-like biological responses, and have a substantially longerduration of biological activity. Therefore, each of these novelprostaglandin analogs is surprisingly and unexpectedly more useful thanone of the corresponding above-mentioned known prostaglandins for atleast one of the pharmacological purposes because it has a different andnarrower spectrum of biological activity than the known prostaglandins,and therefore is more specific in its activity and causes smaller andfewer undesired side effects than when the known prostaglandin is usedfor the same purpose. Moreover, because of its prolonged activity, fewerand smaller doses of the novel prostaglandin analog can frequently beused to attain the desired result.

The novel Formula-V, -VI, and -VII prostaglandin analogs are used asdescribed above in free acid form in ester form, or in pharmacologicallyacceptable salt form. When the ester form is used, the alkyl esters arepreferred, especially the alkyl esters wherein the alkyl moiety containsone to 4 carbon atoms, inclusive. Of those alkyl, methyl and ethyl areespecially preferred for optimum absorption of the compound by the bodyor experimental animal system.

Pharmacologically acceptable salts of these prostaglandin analogs usefulfor the purposes described above are those with pharmacologicallyacceptable metal cations, ammonium, amine cations, or quaternaryammonium cations.

Especially preferred metal cations are those derived from the alkalimetals, e.g., lithium, sodium and potassium and from the alkaline earthmetals, e.g., magnesium and calcium, although cationic forms of othermetals, e.g., aluminum, zinc and iron, are within the scope of thisinvention.

Pharmacologically acceptable amine cations are those derived fromprimary, secondary, or tertiary amines. Examples of suitable amines aremethylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine,triisopropylamine, N-methylhexylamine, decylamine, dodecylarnine,allylamine, crotylamine, cyclopentylamine, dicyclohexylamine,benzylamine, dibenzylamine, a-phenylethylarnine, B-phenylethylamine,ethylenediamine, diethylenetriamine, and like aliphatic, cycloaliphatic,and araliphatic amines containing up to and including about 18 carbonatoms, as well as heterocyclic amines, e.g., piperidine, morpholine,pyrrolidine, piperazine, and lower-alkyl derivatives thereof, e.g.,l-methylpiperidine, 4-ethylmorpholine, l-isopropylpyrrolidine,2-methylpyrrolidine, 1,4- dimethylpiperazine, 2-methylpiperidine, andthe like, as well as amines containing water-solubilizing or hydrophilicgroups, e.g., mono-, di-, and triethanolamine, ethyldiethanolamine,N-butylethanolamine, 2-aminol-butanol, 2-amino-2-ethyl-l,3-propanediol,2-amino- 2-methyll-propanol, tris( hydroxymethyl)arninomethane,N-phenylethanolamine, N-(p-tert-amylphenyl)- diethanolamine,galactamine, N-methylglucamine, N- methylglucosamine, ephedrine,phenylephrine, epinephrine, procaine, and the like.

Examples of suitable pharmacologically acceptable quaternary ammoniumcations are tetramethylammonium, tetraethylammonium,benzyltrimethylammonium, phenyltriethylammonium, and the like.

As discussed above, these novel prostaglandin analogs are administeredin various ways for various purposes; e.g., intravenously,intramuscularly, subcutaneously, orally, intravaginally, rectally,buccally, sublingually, topically, and in the form of sterile implantsfor prolonged action.

9 For intravenous injection or infusion, sterile aqueous isotonicsolutions are preferred. For that purpose, it is preferred because ofincreased water solubility to use the free acid form or thepharmacologically acceptable salt form. For subcutaneous orintramuscular injection, sterile solutions or suspensions of the acid,salt, or ester form in aqueous or non-aqueous media are used. Tablets,capsules, and liquid preparations such as syrups, elixirs, and simplesolutions, with the usual pharmaceutical carriers are used for oral orsublingual administration. For rectal or vaginal administration,suppositories, tampons, ring devices, and preparations adapted togenerate sprays or foams or to be used for lavage, all prepared as knownin the art, are used. For tissue implants, a steriletablet or siliconerubber capsule or other object containing or. impregnated withthesubstanceisused. l

vThe novel compounds of Formulas V, VI and VII wherein R is other thanhydrogem, i.e.,: the esters wherein R is alkyl of one to 8 carbon atoms,inclusive,

cycloalkyl of 3 to 10 carbon atoms, inclusive, aralkyl of '7 .to. 12.carbon atoms, inclusive, phenyl, or phenyl substituted with l to 3chloro or alkyl of l to -4 carbon atoms, inclusive, are prepared-fromthe corresponding ,acidslof Formulas V, VI, and VII; i.e., wherein R ishydrogen, by methodsknown in the art. For example,

.the alkyl, cycloalkyLand aralkyl esters are prepared by interaction ofsaid acids with the appropriatediazohydrocarbon. For example, when,diazomethane is used,

the methyl esters are produced. Similar use of diazoethane, diazobutane,l-diazo-2-ethylhexane, diazocyclohexane, and phenyldiazomethane, forexample, gives. the ethyl, butyl, 2-ethylhexyl, cyclohexyl, and

benzyl esters, respectively.

i'Esterification with diazohydrocarbons is carried out by mixing asolution of the diazohydrocarbon in a suit- -.-able inert solvent,preferably diethyl ether, with the acid reactant, advantageously-in thesame or a different inert diluent. After the esterification reaction iscomplete, the solvent is removed by evaporation, and the -.esterpurified if desired by conventional methods, prefpounds of Formulas V,VI, and VII comprises transformation of the free acidto thecorrespondingsilver salt,

followed by interaction of that salt with an alkyl iodide. Examples ofsuitable iodides are methyl iodide, ethyl iodide, butyl iodide, isobutyliodide, tert-butyl iodide,

cyclopropyl iodide, cyclopentyl iodide, benzyl iodide,

, phenethyl iodide, and the like. The silver salts are prepared byconventional methods, for example, by dis-' solving the acid in colddilute aqueous ammonia, evaporating the excess ammonia at reducedpressure, and then adding the stoichiometric amountof silver nitrate.

The phenyl and substitutedphenyl esters 'of the For- I mula-V, '-VI, and,-VII. compounds are prepared by silylating the acid to protectthehydroxy groups, for example, replacing each 'OH with ,O--Si(Cl-l Doingthat may also change COOl-I to.COO-Si- (CH A brief treatment of thesilylated compound with water will change COOSi(CI-l back to -COOH.Procedures for this silylat'ion are known in the art and are discussedhereinafter. Then, treatment of the silylated compound with oxalylchloride gives the acid chloride which is reacted with phenol or theappropriate substituted phenol to give a silylated phenyl or substitutedphenyl ester. Then the silyl groups, e.g., -OSi(CI-l are changed back to-OI-l by treatment with dilute acetic acid. Procedures for thesetransformations are known in the art.

The novel Formula-V, -VI, and -VII acids (R is hydrogen) are transformedto pharmacologically acceptable salts by neutralization with appropriateamounts of the corresponding inorganic or organic base, examples ofwhich correspond to the cations and amines listed above. Thesetransformations are carried out. by a variety of procedures known invthe art to be generally useful forrthe preparation of inorganic, i.e.,metal or ammonium salts, amine acid addition salts, and quaternaryammonium salts. The choice of procedure depends in part uponthesolubility characteristics of the particularsalt to be prepared. In thecase of the inorganic salts, it is usually suitable to dissolve the acidin water containing the stoichiometric amount of a hydroxide, carbonate,or bicarbonate corresponding to the inorganic salt desired. For,example, such use of sodium hydroxide,sodium carbonate, or sodiumbicarbonate gives a solution of the sodium salt of the prostanoic acidderivative. Evaporation of the water or addition of'a watermisciblesolvent of moderate polarcorresponding to the desired cation is thenadded to that solution. If the resulting salt does not precipitate, it

is usually obtained in solid form by addition of a miscible diluent oflow polarity or by evaporation. If the amine is relatively volatile, anyexcess can easily be removed byevaporation. It is preferred'to usestoichiometric amounts of the less volatile amines.

Salts wherein the cation is quaternary ammonium are produced by mixingthe acid with the stoichiometric amount of the corresponding quaternaryammonium hydroxide in water solution, followed by evaporation of -thewater.

The novel,.compounds of Formulas V, VI, and VII .wherein R is hydrogenor methyl, i.e., the free acids Vlla WQORE H Ra In Formulas VIIa, VIII,and IX, R is either hydrogen or methyl, and R is hydrogen or acetyl.

It will be observed that the compounds encompassed by Formula VIIa arealso encompassed by VII. Thus, some Formula-VII compounds are usefulboth as intermediates and for pharmacological purposes.

These Formula-VIIa, VIII, and IX starting materials are all derivativesof prostanoic acid. The compounds of Formula VIII are known in the artor are available by methods known in the art. See, for example,Weinheimer et al., Tetrahedron Letters, No. 59, 5185 (1969); H. W.Youngken, Jr. (ed), Food-Drugs from the Sea, Proc. Marine TechnologySociety, pp 311-314 (1969). The Formula-VIII compound wherein R and Rare both hydrogen is designated l5B-PGA alternatively l5(R)-PGA orl5-epi-PGA The other compounds encompassed by Formula VIII aredesignated l5/3-PGA acetate, l5B-PGA methyl ester, and ISB-PGA acetatemethyl ester.

The compounds of Formula VIIa are new in the art and methods forpreparing them are described hereinafter. The F ormula-Vlla compoundwherein R and R are both hydrogen is designated l5B-PGE alternatively(R)-PGE or IS-epi-PGE The other compounds encompassed by Formula VII aredesignated 15B-PGE IS-acetate, 15B-PGE methyl ester, and l5B-PGE acetatemethyl ester.

The compounds of Formula IX are known in the art.

' See, for example, British Specification No. 1,097,533.

Novel methods for preparing these Formula-IX compounds are describedhereinafter. The Formula-IX compound wherein R and R are hydrogen isdesignated PGA The other compounds encompassed by Formula IX aredesignated PGA acetate, PGA methyl ester, and PGA acetate methyl ester.

All of the compounds of Fonnulas VIIa, VIII, and IX are obtained byextraction from a marine invertebrate. The compounds of Formula VIIa andVIII, i.e., the 153 compounds, are obtained from colonies of Plexaurahomomalla (Esper), 1792, forma R. The compounds of Formula IX, i.e., the15(5) or alpha compounds, are obtained from colonies of Plexaurahomomalla (Esper I792, forma S.

These Plexaura homomalla forms are members of the Holaxo'nia, familyPlexauridae, genue Plexaura. See, for example, Bayer, The Shallow-WaterOctocorallia of the West Indian Region, Martinus Nijhoff, The Hague(1961). Colonies of these Plexaura homomalla forms are abundant on theocean reefs in the zone from the low-tide line to about 25 fathoms inthe tropical and subtropical regions of the western part of the AtlanticOcean, from Bermuda to the reefs of Brazil, including the eastern shorereefs of Florida, the Caribbean island VIII and mainland reefs, and theGulf of Mexico island and mainland reefs. These colonies are bush-likeor small tree-like in habit, and are readily identified for collectionas Plexaura homomalla (Esper), 1792, by those of ordinary skill in thisart. Forms R and S are distinguished by the methods described inPreparation 1 below.

The colonies of these two forms of Plexaura homomalla are easilyseparated into an outer bark-like cortex and an inner wiry proteinaceousstem or skeleton. Symbiotic algae or Zooxanthellae are also present inthe colonies. Weinheimer et al., cited above, disclose the occurence ofthe Formula-VIII compounds wherein R and R are both hydrogen and whereinR is methyl and R is acetyl in the air dried cortex of Plexaurahomomalla (Esper).

The choice of isolation or extraction method is determined by theparticular Formula-VIIa, VIII, or IX compound desired. Maximum yield ofthe Formula- VIII or lX diester is realized by freezing whole orcoarsely cut or chopped fresh Plexaura homomalla colonies within an hourand preferably sooner after the colonies are removed from the reef. Forsmall scale collections, this freezing is done advantageously bycontacting the colonies or pieces with solid carbon dioxide. For largerscale collections, other suitable freezing methods are known to the art.The frozen colonies or colony pieces should be kept frozen, preferablybelow about 20C. until the extraction takes place.

The major component of fresh Plexaura homomalla (Esper), I792, forma Ris l5B-PGA acetate methyl ester, the Formula-VIII compound wherein R2 ismethyl and R is acetyl. Relatively minor components are the hydroxymethyl ester, the acetate, and the hydroxy acid of Formula VIII and theISB-PGEz compounds encompassed by Formula VIIa. Of the latter, the15B-PGE2 acetate methyl ester (R2 is methyl and R is acetyl) is the mostabundant. The major component of Plexaura homomalla (Esper), 1792, formaS is PGA acetate methyl ester, the Formula-IX compound wherein R ismehtyl and R is acetyl. Relatively minor components are the hydroxymethyl ester, the acetate, and the hydroxy acid of ForsubclassOctocorallia, order Gorgonacea, suborder l IX, d the PGE compoundscorresponding to high polarity, e.g., dichloromethane or methanol,advantageously, for 15 to 30 minutes in a high speed mixer. The desiredcompounds are isolated from the extract by evaporation, and thenchromatography of the resulting residue. By this procedure, about 24 g.of ISB-PGA acetate methyl ester, and about one g. each of lB-PGA methylester and ISB-PGE are obtained by dichloromethane extraction of I500 g.of frozen Plexaura homomalla (Esper), 1792, forma R colonies or colonypieces. Similarly, relatively large amounts of PGA acetate methyl esterare obtained from frozen Plexaura homomalla (Esper), I792, forma S.

When the IS-hydroxy methyl ester of Formula Vlla, VIII, or IX (R ismethyl, R is hydrogen) is desired as a starting material, a suitablemethod comprises grinding the frozen whole Plexaura homomalla coloniesor colony pieces as above, and then contacting the resulting particleswith a lower alkanol, preferably methanol or ethanol, at 25C. forseveral days. The solvent is then evaporated and the residuechromatographed to give substantially larger amounts of the hydroxymethyl ester compound to the acetate methyl ester compound. When thecontact between the Plexaura homomalla particles and the alkanol issubstantially shorter, substantially the same amount and ratio of thevarious Formula-Vlla, -VIII, or -IX compounds is obtained with thealkanol as with dichloromethane. An alterna-. tive method for obtainingthese IS-hydroxy methyl esters is described below.

When l5B-PGA l5B-PGE or PGA (R and R in Formulas Vlla, VIII, and IX areboth hydrogen) are desired as starting materials in the novel processesof this invention, they are prepared from the correspondding methylesters and IS-acetate methyl esters after those have been extracted fromthe Plexaura homomalla colonies or colony pieces as described above. Asuitable method for removing the acetyl group of each of theFormula-Vlla, -VIII, and -IX IS-acetate methyl esters comprises mixingthe acetate methyl ester in lower alkanol solution, preferably inmethanol solution, with a strong acid, e.g., perchloric acid, for abouthours at C. A suitable method for removing the methyl group of any ofthe Formula-Vlla, -VIII, and -IX methyl esters is the enzymatichydrolysis described in West Germany Offenlegungschrift No. 1,937,912,reprinted in Farmdoc Complete Specifications, Book No. 14, No. 6869R,Week R March 18, 1970.

Another method for obtaining l5B-PGA lSB-PGE or PGA from Plexaurahomomalla colonies or colony pieces comprises freezing the Plexaurahomomalla colonies or colony pieces, preferably at a temperature belowabout -20C., and then allowing the colonies or colony pieces to thaw andwarm to a temperature in the range 20 to C. The thawed colonies orcolony pieces are then.maintained in the range 20 to 30 C. for at least24 hours. After that treatment, substantially none of the Formula-Vlla,-VIII, and -IX compounds wherein R is methyl and R is acetyl arepresent, the

principal Formula-Vlla, -VIII, and -IX compounds present being thosewherein R and R are both hydrogen, the minor components being thosewherein R is methyl and R is hydrogen or wherein R2 is hydrogen and R isacetyl. As before, Formula-Vlla and -VIII compounds are obtained fromcolonies of Plexaura homomalla (Esper), 1792, forma R, and Formula-IXcompounds are obtained from colonies of Plexaura homomalla (Esper),1792, forma S.

A preferred procedure for the PGA and PGE type free acids comprisesgringing the Plexaura homomalla colonies or colony pieces, preferably toa particle size with the largest dimension about 5 mm., and thenmaintaining the mixture in contact with water at a temperature in therange 20 to 30 C. for at least 24 hours. This mixture is filtered, andthe filtrate is extracted with an appropriate water-immiscible solvent,e.g., ethyl acetate. The solid residue is also extracted with anappropriate solvent, e.g., methanol. The two extracts are evaporated,and the total residue is chromatographed to give Formula-Vlla and -VIIIor Formula-IX compounds, the principal component in each case being thecompound wherein R and R are both hydrogen.

Since our invention of the novel processes for transforming PGA and15B-PGA and their methyl esters and acetate methyl esters to the variousprostanoic acids and esters disclosed herein, it has now been found thatsmall amounts of the 5,6-trans compounds of PGA and l5B-PGA and theirmethyl esters and acetate methyl esters are also obtained from Plexaurahomomalla (Esper), 1792, forms R and S. These 5,6- Trans compounds areextracted with and accompany the corresponding PGA -type compoundsthrough many of their transformations. For example, PGA containing5,6-trans-PGA yields a mixture of PGE and 5,6-trans-PGE by the processrepresented in Chart E below.

When it is desired, for pharmacological purposes, to prepare the majorproducts of this invention free of 5,6-trans compounds, those 5,6-transcompounds are separated either from the starting reactants or from theproducts. In either case, several methods are available forseparating'the 5,6-trans-PG compounds from the PG compounds. One methodis by means of a silversaturated ion-exchange resin (for example, see E.A. Emken et al., J. Am. Oil Chemists Soc. 41, 388 (1964)), illustratedbelow in Preparations 5 and 6. The other methodis by preferentiallyforming a mercuric acetate adduct of the 5,6-cis compound which isextractable into polar solvents illustrated below in Preparation 7.

Following the processes discussed herein and the procedures of theExamples below, the 5,6-trans-PG (and -l5B-PG compounds are transformedto other 5,6-trans-PG (and -l5B-PG compounds, e.g., 5,6- trans-PGA to 5,6-trans-PGE 5,6-trans-l5B-PGA acetate methyl ester to 5,6-trans-l5B-PGFa acetate methyl ester, and the like.

As mentionedabove, the Formula-Vlla, -VIII, and -IX compounds arestarting materials for the preparation of PGE PGF a and PGF B the methylesters of those, and also the novel compounds of Formulas V and VI, andsome of the novel compounds of Formula VII. The novel processes usingthese starting materials will now be described.

The Formula-VIII and -IX starting materials are both of the PGA-type.According to the novel processes of this invention, those are firsttransformed to corresponding PGE-type compounds. The chemicalreacwherein R is hydrogen, acetyl, or Si(A) when R, 5 is hydrogen ormethyl, and R is SI(A) when R is SI-(A) R is hydrogen or methyl; and Bis wherein A is as defined above. In Formula XI, of Chart A,

Thus, Formula X in Chart A encompasses the starting materials ofFormulas VIII and IX obtained from indicates attachment of the epoxyoxygen to the ring in Plexaura homomalla, and also compounds of theforalpha or beta configuration. In Formulas XII and XIII mula: 15 ofChart A, indicates attachmentt of hydroxy to the wherein R is as definedabove, and Z is ring in alpha or beta configuration.

CHART A (reduction) 'W COORZ XH Mi -continued (hydrolysis) It will beobserved in Chart A that the Formula-XII and XIII products eachencompass four stereoisomeric groups of compounds. Included arecompounds with the l1oz,l5(S) configuration of PGE (Formula II, above),compounds with the configuration of 1 101,158- PGE (Formula Vlla) asobtained from Plexaura homomalla (Esper), 1792, forrna R, and both the15(8) and 15(R) compounds with the 11/3 configuration of the novelFormula-V compounds of this invention wherein Y is i.e., llB-PGE andIIBJSBPGE If the Formula-XII or -Xll1 product is to have the 15(8)configuration, e.g., PGE or llB-PGE then the Formula-X starting materialshould have the 15(5) configuration, i.e., G

shouldbe If a 153 compound of Formula X1] or XIII is desired, e.g.,15'B-PGE or llB,lBPGE ,then the Formula-X starting material should havethe (R) or 15-epi configuration, i.e., G should be 'or'bromide, e.g.,p-toluenesulfonyl chloride. This reaction is done in the presence of atleast sufficient tertiary ride or hydrogen bromide by-product, and at alow temperature, preferably'in the range .l5 to +l5 C. The presence ofan inert liquid diluent, e.g., tetrahydrofuran, is helpful to maintain amobile homogenous reaction mixture. At 0 C. and with methanesulfonylchloride, usually to 60 minutes is a sufficient reaction time. Theproduct is hydrolyzed to a mixture of Xlll amine, e. g., triethylamine,to absorb the hydrogen chlo- 15(S) (alpha) and 15(R) hydroxy compounds.These are separated by procedures known in the art, and the 15(S)product is purified by procedures known in the art, advantageously bychromatography on silica gel. This reaction is also used to transform15(S) Formula- IX starting materials wherein R is hydrogen or methyl andR is hydrogen to the corresponding 15(R) compounds. In each case, amixture of 15(R) product and 15(S) starting material is obtained, thecomponents of which are separated as described above.

Another method of transforming a ISB-PG compound to a PG compound is byconverting it to a mixture of PG l5-formate and ISB-PG l5-formatecompounds, separating the PG l5-formate, and hydrolyzing the PG 151formate to the desired PG compound (see J. -E. Pike et al., J. Org. Chem34, 3552 (1969)).

The mixture of alpha and beta lS-formates is prepared by maintaining the156 compound, e.g. 15B- PGE 15B-PGA or 15,8-PGF a in formic acidbuffered with an alkali metal formate in the range 10 to 50 C. until asubstantial amount of the 158 compound, e.g. ISB-PGE lS-formate, hasbeen transformed to the PG IS-formate. The mixture of the PG 15-formateand 15B 15-formates thus obtained is then separated by known methods,e.g. by chromatography.

The PG 15-formate can then be hydrolyzed to the desired PG l5-hydroxycompound. The ISBPG l5formate yields the corresponding 15,8-PG15-hydroxy compound, which is then recycled through the above steps forfurther isomerization to the'PG compound if desired.

This procedure is also useful to transform a PG compound to a 1513 PGcompound, by obtaining a mixture of the intermediate PG l5-formate andISB-PG 15-formate compounds, separating them, and hydrolyzing them tothe respective PG 15-hydroxy and ISB-PG l5-hydroxy compounds. In thiscase, the PG compound is recycled for further isomerization to the 153compound.

Referring again to Chart A, the transformation of starting material X toepoxide X1 is carried out by reacting X with any agent known toepoxidize an aB- unsaturated ketone without reacting with isolatedcarbon-carbon double bonds, for example see Steroid Reactions, CarlDjerassi, ed., Holden-Day Inc., 1963, p. 593. Especially preferred areaqueous hydrogen peroxide or an organic tertiary hydroperoxideSee, forexample, Organic Peroxides, A. V. Tobolsky et al., IntersciencePublishers, N.Y., 1954. For this purpose, the peroxide or hydroperoxideis employed in an amount of at least one equivalent per mole of Formula-X reactant in the presence of a strong base, e.g., an alkali metalhydroxide, a metal alkoxide, or a quaternary ammonium hydroxide. Forexample, there is employed lithium hydroxide, sodium hydroxide,potassium hydroxide, lithium ethoxide, lithium octyloxide, magnesiummethoxide, magnesium isopropoxide, benzyltrimethylammonium hydroxide,tetraethylammonium hydroxide, butyltrimethylammonium hydroxide,butyldiethylphenylammonium hydroxide, benzylethyldimethylammoniumhydroxide, benzyldimethyloctadecylammonium hydroxide,benzyldodecyldimethylammonium hydroxide, decyldimethyylphenylammoniumhydroxide, and the like. See, for example, Sidgwick, Organic Chemistryof Nitrogen, Third Edition, rev. by Miller and Springall, Oxford, 1966,pp. 116-127.

The ratio of alpha to beta epoxide formed in the reaction is related tofour factors: the epoxidizing agent, the base, the diluent, and thetemperature. Hydrogen peroxide is employed in the concentrations usuallyavailable, for example 3 to 90%, although 30% is especially convenient.When the alpha epoxide is the desired product, tert-bytyl hyydroperoxideis especially preferred as the epoxidizing agent. Examples of otherorganic tertiary hydroperoxides useful for this purpose are tert-pentylhydroperoxide, decahydronaphthyl hydroperoxide, a,a-dimethylbenzylhydroperoxide, and 1,1-diphenylethyl hydroperoxide. The base is presentin the proportion of 0.1-3.0, preferably about 0.1-0.5 equivalent ofbase per mole of starting material X when R,, is methyl and R is acetyl;preferably about 1.5-2.5 equivalent of base per mole of startingmaterial VIII or IX wherein R and R are hydrogen. When the alpha epoxideis the desired product, lithium hydroxide, lithium or magnesiumalkoxides of one to 8 carbon atoms, and benzyltrimethylammoniumhydroxide are the preferred bases, although the lithium and magnesiumcompounds are especially preferred.

It is advantageous to use an inert liquid diluent in the epoxidationstep to produce a mobile homogenous reaction mixture, for example, alower alkanol, dioxane, tetrahydrofuran, dimethoxyethane,dimethylsulfoxide, or dimethyl- When the alpha epoxide is preferred,tetrahydrofuran or the less polar dimethoxyethane are especiallypreferred as the diluent. A reaction temperature in the range 60 to 0 C.is generally preferred, especially below -10 C. The lower temperaturesbelow 30 C. are especially preferred for favoring formation of alphaepoxide over beta epoxide. At a temperature of 20 C., the epoxidation isusually complete in 3 to 6 hours. It is also preferred that the reactionbe carried out in an atmosphere of an inert gas, e.g., nitrogen, helium,or argon. When the reaction is complete as shown by the absence ofstarting material on TLC plates (3% acetone in dichloromethane), thereaction mixture is neutralized, and the epoxy product is isolated byprocedures known in the art, for example, evaporation of the diluent andextraction of the residue with an appropriate water-immiscible solvent,e.g., ethyl acetate.

This transformation of X to XI usually produces a mixture of Formula-XIalpha and beta epoxides both with either the (R) or 15(S) configurationdepending on the configuration at C-l5,in the Formula-X startingmaterial. Although these mixtures are separated into the individualalpha and beta isomers, for example, by chromatography by proceduresknown to be useful for separating alpha and beta epoxide mixtures, it isusually advantageous to transform the Formula-XI mixture of alpha andbeta epoxides to the wherein R is hydrogen, acetyl, or Si(A) wherein Ais as defined above. For either definition of G, i.e., R configurationor S configuration, when R, is hydrogen, more Formula-XI beta epoxide isformed than when R is acetyl, and more Formula-XI beta epoxide is formedwhen R is acetyl than when R is Si-(A) For example, when G in formula Xis the preferred basic hydrogen peroxide epoxidation gives about equalamounts of alpha and beta epoxides, but when G in formula X is H OCOCHabout 3 parts of alpha epoxide and 1 part of beta epoxide are obtained,and when G in formula X is H OSi(CH about 4 parts of alpha epoxide andone part of beta epoxide are obtained, both reactions with the sameepoxidation reagent. When G in formula X is about 1 part of alphaepoxide and 3 parts of beta epoxide are obtained with basic hydrogenperoxide, but when G is about 6 parts of alpha epoxide and 4 parts ofbeta epoxide are obtained with the same epoxidation reagent.

Each of the novel Formula-V and -VI compounds of this invention has ahydroxy attached in beta configuration to the cyclopentane ring. Some ofthe compounds of Formula V, i.e., when Y is I are encompassed by FormulaXll (Chart A). The other Formula-V compounds and all of the Formula Vlcom- 21 pounds are prepared as described below from Formula- XIIcompounds wherein the hydroxy at C-ll is attached in beta configuration.Therefore, when a Formula-V or Formula-VI compound is described as afinal product for pharmacological purposes, there is advantage inchoosing a corresponding Formula-X starting material which gives themaximum amount of beta epoxide during the transformation of X to XI.Those will be the Formula-X compounds wherein G is H OH or H OH.

On the other hand, when a prostanoic acid product with the natural Ilaconfiguration for the hydroxy at 011 is the desired final product, e.g.,PGE PGF a or PGF B there is advantage in choosing a Formula- X startingmaterial which gives a greater amount of the alpha epoxide during thetransformation of X to XI. Those would be Formula-X compounds wherein Xis or the corresponding 15(S) compounds.

As mentioned above, the starting materials of Formula X encompass notonly the Formula-VIII and IX compounds obtained from Plexaura homomallabut also the silyl compounds of Formula X When desired as reactants,these 'silyl compounds are prepared by silylation of PGA lB-PGA or themethyl esters of those. These silylations are carried out by proceduresknown in the art. See, for example, Pierce, Silylation of OrganicCompounds, Pierce Chemical Co., Rockford, Ill. (1968). The C-l5 hydroxygroup of PGA /3-PGA or, their methyl esters is transformed to an O-Si(A)moiety wherein A is as defined above, sufficient silylating agent being1 used according to known procedures to accomplish that. The necessarysilylating agents for this purpose are known in the art or are preparedby methods known in the art. See, for example, Post, Silicones and OtherOrganic Silicon Compounds, Reinhold Publishing Corp., New York, NY.(1949). In the case of PGA and l5B-PGA excess silylating agent andprolonged treatment also transform the COOH' to COO-Si(A) It is optionalin transforming X to XI whether or not this COOH of PGA or l5/3-PGA isesterified to COOSi--'(A) since that ester group is transformed toCOOI-I during formation and isolation of the Formula-XI epoxide product.

The various As of a Si-(A) are alike or different. For example, anSi-(A) can be trimethylsilyl, dimethylphenylsilyl, ormethylphenylbenzylsilyl.

When it is desired to retain the Si-(A) moiety at C-l5 in the Formula-XIepoxide product, for example, to give steric control in a subsequentreaction, it is important in isolating the epoxide that the presence ofacid be avoided and that contact with water be minimized unless thewater is kept cold, i.e., below about 10 C.

Referring again to Chart A, the transformation of epoxide XI to hydroxycompound XII is accomplished by reduction with chromium (Il) salts,e.g., chromium (II) chloride or chromium (II) acetate. Those salts areprepared by methods known in the art, e.g., Inorganic Syntheses, VIII,(1966); ibid., VI, 144 (I960); ibid. III, 148 (1950); ibid. I, 122(1939); and references cited in those. This reduction is carried out byprocedures known in the art for using chromium (II) salts to reduceepoxides of (JAB-unsaturated ketones to B-hydroxy ketones. See, forexample, Cole et al., J. Org. Chem. 19, 131 (1954), and Neher et al.,Helv. Chem. Acta 42, 132 (1959). In these reactions, the absence of airand strong acids is desirable. If it is desired to maintain a Si(A)moiety on C-l5, a neutral reaction mixture is preferred. An especiallypreferred procedure is to generate the chromium (ll) ion in the presenceof the Formula-XI epoxide, for example, by mixing the epoxide with achromium (Ill) salt, e. g., the chloride, with metallic zinc in thepresence of acetic acid. The desired Formula-XII compound is isolatedfrom the reduction reaction mixture by methods known in the art, carebeing taken to minimize contact of the product with acid and water,especially warm water, when retention of a -Si( A) at C-l5 is desired.

Unexpectedly, amalgamated aluminum metal has also been foundto be usefulas a reducing agent in place of chromium (II) salts to transform FormulaXI epoxides to Formula XII hydroxy compounds. This reagent is previouslynot known to be useful for this type of reaction; This use ofamalgamated aluminum represents a distinct and separate aspect of thisinvention.

Amalgamated aluminum is prepared by procedures known in the art, forexample, by contacting aluminum metal in the form of foil, thin sheet,tumings, or granules with a mercury (II) salt, for example, mercuricchloride, advantageously in the presence of sufficient water to dissolvethe mercury (II) salt. Preferably, the surface of the aluminum metal isfree of oxide. That is readily accomplished by physical removal of theusual oxide layer, e.g., by abrasion or scraping, or chemically, e.g.,by-etching with aqueous sodiumhydroxide solution. It is only necessarythat the aluminum surface be amalgamated. The amalgamated aluminumshould be freshly prepared, and maintained in the absence of air andmoisture until used.

The reductive opening of the Formula-XI epoxide ring is accomplished bycontacting said epoxide with the amalgamated aluminum in the presence ofa hydroxylic solvent and sufficient inert organic liquid diluent to givea mobileand homogeneous reaction mixture with respect to the hydroxylicsolvent and said epoxide. Among hydroxylic solvents, water is especiallypreferred although lower alkanols, e.g., methanol and ethanols are alsooperable.

Examples of inert organic liquid diluents are nor mally liquid etherssuch as diethyl ether, tetrahydrofuran, dimethoxyethane, diglyme(dimethyl ether of diethylene glycol), and the like. Especiallypreferred is tetrahydrofuran. When a water-immiscible liquid diluent isused, a mixture of water and methanol or ethanol is especially useful inthis reaction since the latter two solvents also aid in forming thedesired homogeneous reaction mixture. For example, a mixture of diethylether and water is used with sufficient methanol to give a homogeneousreaction mixture.

This reductive opening requires two hydrogen atoms per molecule ofepoxide. Amalgamated aluminum reacts readily with water and more slowlywith other hydroxylic solvents to give hydrogen. One atomic equivalentof aluminum required 3 molecular equiva- 23 lents of the hydroxylicsolvent to give 3 atomic equivalents of hydrogen. Therefore, onemolecular equivalent of epoxide requires two-thirds atomic equivalent ofaluminum and 2 molecular equivalents of the hydroxylic solvent.Evolution of hydrogen gas (H molecules) is observed during thisreductive opening of the epoxide. It is not known whether the reductiveopening is caused by hydrogen atoms or hydrogen molecules. However, someof the hydrogen gas escapes from the reaction mixture. Therefore, it ispreferred to use an excess of amalgamated aluminum and hydroxylicsolvent, preferably at least one atomic equivalent of aluminum and 3molecular equivalents of hydroxylic solvent per molecular equivalent ofepoxide. Because of the relatively high economic value of the epoxidecompared with amalgamated aluminum and hydroxylic solvents, it ispreferred to assure maximum yields of the desired Formula-XII hydroxycompound by use of substantially greater excess of amalgamated aluminumand hydroxylic solvent, e.g., up to times or more of those reagents thanis theoretically required.

The reductive opening of the epoxide is carried out by mixing a solutionof the epoxide in the organic diluent with the amalgamated aluminum andthe hydroxylic solvent. Since the reaction is exothermic, it is usuallyadvantageous to cool the solution to a low temperature, e.g., C. to 0C., before adding the amalgamated aluminum and hydroxylic solvent and tomaintain the reaction mixture in the range 20 to 30 C. by externalcooling. This is especially advantageous when water is used as thehydroxylic solvent. Higher reaction temperatures are operable but notpreferred when a high yield of the Formula-XII products is desired.Stirring is preferred during the reaction since the reaction mixture isheterogeneous with respect to the solution and the amalgamated aluminum.

For reasons not understood, better yields and a shorter reaction timeare usually observed when only part of the amalgamated aluminum is addedat the start of the reaction, additional portions being added during thereaction, e.g., at l-hour intervals, than when the entire amount ofamalgamated aluminum is added at the start of the reaction. A suitableprocedure is to add about one-third of the amalgamated aluminum at thestart, about one-third after one hour, and another third after a secondhour. The course of the reaction is advantageously determined bywithdrawing small portions of the solution and determining the presenceor absence of starting material by thin layer chromatography. Forexample, when R is methyl and G is H OCOCH;

in Formulas XI and XII, a suitable TLC system is ethylacetatecyclohexane-acetic acid (40/60/2), the Formula-XI startingmaterial having r, 0.64, and the two Formula-XII products having r, 0.25(H3) and r; 0.20 (Ila).

As a modification of the above-described process for reductive openingof the epoxide, it has been found quite unexpectedly that instead ofemploying a Formula-XI compound wherein R is hydrogen, the reductiveopening reaction proceeds more smoothly and completely if there is used,instead, an epoxide of the formula Xla wherein G and are as definedabove and R is a cation of an alkali or alkaline earth metal or aquaternary ammonium group.

Thus, the Formula-XI epoxide compound is treated with a hydroxide oroxide of lithium, sodium, potassium, magnesium, calcium, barium, orstrontium prior to contacting with the aluminum amalgam. Optionally, thequaternary ammonium bases are used for this neutralization, for examplebenzyltrimethylammonium hydroxide. The base is used in equivalent amountto the acid so that R is replaced by the corresponding metal orquaternary ammonium cation. Alternatively, instead of the hydroxides oroxides, there are employed the hydrides, the carbonate, thebicarbonates, or the alkoxides, for example lithium hydride, potassiumcarbonate, sodium bicarbonate, magnesium methoxide, and the like, whichform the corresponding Formula-XIa salts with the Formula-XI free acid.Alternatively, a metal or quaternary-ammonium carboxylate compound orFormula-Xla salt carried forward from the epoxidation step, whetherisolated in that step or not, is employed in the reductive step withaluminum amalgam. It is preferred that the Formula-Xla salt be solublein the organic diluent-alkanolwater or organic diluentwater medium usedfor the reduction step. By using the above-described salts, thereduction step proceeds smoothly without formation of insoluble aluminumsalts which hinder the reaction. Following the reduction or hydrolysisstep, the R cations are replaced with hydrogen by means known in theart, for example by acidification and extraction of the acid compoundinto an organic phase.

The desired Formula-XII hydroxy products are isolated by filtration ofthe reaction mixture, advantageously after addition of magnesium sulfateas a filter aid, and evaporation of the organic diluents. TheFormula-XII products are then hydrolyzed if desired to remove -Si(A)from C-l5, and the Ila and IIB products of Formula XIII are separated,if desired, by procedures known in the art, e.g., chromatography onsilica gel.

The products of FormulaXlI are all of the PGE -type and include PGE PGE15-acetate, PGE methyl es: ter, PGE lS-acetate methyl ester, PGE and PGEmethyl ester with an -OSi-(A) at C- l 5, the corresponding 15,8compounds, and compounds corresponding to all of those wherein hydroxyis attached to G1] in beta configuration.

As mentioned above, the transformation of X to XI to XII usually gives amixture of Formula-XII PGE-type products, part with alpha and part withbeta configuration for the hydroxy at C-1 1. There are severalaltematives regarding that mixture. If OSi(A) is attached to C-15, thatcan readily be transformed by hydrolysis to OH. These hydrolyses arecarried out by prior art procedures known to be useful for transformingsilyl ethers to alcohols. See, for example, Pierce, cited above,especially p. 447 thereof. A mixture of water and sufficient of awater-miscible organic diluent to give a homogeneous hydrolysis reactionmixture represents a suitable reaction medium. Addition of a catalyticamount of an organic or inorganic acid hastens the hydrolysis. Thelength of time required for the hydrolysis is determined in part by thehydrolysis temperature. With a mixture of water and methanol at 25 C.,several hours is usually sufficient for hydrolysis. At C., several daysis usually necessary. Also, if O- COCH is attached to C-1 5, that canreadily be transformed to OI-I by acid-catalyzed alcoholysis asdescribed above for removing the acetyl group of the Formula-VIII and-IX PGA-type starting materials. Both of those transformations are shownin Chart A, i.e., XII to XIII. Either before or after thosetransformations of XII to XIII, the Formula-XII or -XIII mixture of Ilaand [I3 isomers can be separated by methods known in the art,advantageously by chromatography on silica gel.

Further regarding the Formula-XIII compounds, those compounds whereinthe configuration of the hydroxy at C11 is beta are within the scope ofthe Formula-V novel compounds of this invention, and those compoundswherein the configuration of the hydroxy at C-ll is alpha and B is arewithin the scope of the Formula-VII novel compounds of this invention.Both groups of novel compounds are used for pharmacological purposesdescribed above for those compounds, the acids also being useful asreactants to prepare pharmacologically usefuljesters andpharmacologically acceptable 'and useful'salts, bothas described above.Moreover, Formula-XIII compounds wherein the configuration of thehydroxy at C-1 1 is alpha and B is are and methyl ester, both of knownpharmacological utility.

Still further regarding the separated compounds of Formulas Xll andXlIl, when a compound with one configuration at C-1 1, either alpha orbeta, is desired as an intermediate-or forpharmacological purposes, theother isomer is readily dehydrated to give additional Formula-X PGA-typestarting material which is then used as a starting material according tothe processes defined in Chart A and procedures described above to giveadditional of the desired isomer. These dehydrations are accomplished byprocedures known in the art for dehydration of PGE-type compounds toPGA-type compounds. See, for example, Pike et al., Proc. Nobel SymposiumII, Stockholm (1966), Interscience Publishers, New York, p. l62 (1967),and British Patent Specification No. 1,097,533. These are acidicdehydrations, and alkanoic acids of 2 to 6 carbon atoms, inclusive,especially acetic acid, are preferred for this purpose. Dilute aqueoussolutions of mineral acid, e.g., hydrochloric acid, especially in thepresence of a solubilizing diluent, e.g., tetrahydrofuran, are alsouseful as reagents for these acidic dehydrations, although thesereagents may also cause partial hydrolysis of the Formula-XII or -XIIImethyl esters to carboxylic acids. A Si(A) moiety at C15 is also removedduring all of these acidic dehydrations.

Still further regarding the Formula-XII and -XIII compounds, either asmixtures or separately, any of those is transformed to other usefulcompounds or mixtures by changing these PGE-type compounds to PGF-typeproducts by reducing the ring carbonyl at C-9 to alpha hydroxy or betahydroxy. Those transformations are shown in Chart B.

In Chart B, R is hydrogen, methyl, or Si(A) R is hydrogen or methyl, Ris hydrogen or Si(A) and G is H on, or H 0R,

H OH,

H OH or and indicates attachment to the cyclopentane ring in alpha orbeta position.

CHART B I Xll -continued (silylation) (hydrolysis or alcoholysis) lB-PGEacetate, ISB-PGE methyl ester, and B- PGE methyl ester acetate areobtained from Plexaura homomalla (Esper), 1792, forma R. All of thosecompounds are encompassed by Formula XII, and thus, extraction of thisform of Plexaura homomalla provides an alternative source of thesestarting materials.

Referring to Chart B, the starting material XII can be a mixture ofcompounds with regard to the configuration of C-] l, or the startingmaterial can be stereochemically pure with respect to C-1 1, dependingupon whether there has been an earlier separation of Ila and H3 isomers(see above discussion of Chart A reactions).

The transformation of PGE-type starting material XII to PGF-type productXVI involves reduction of a ring carbonyl to a ring hydroxy. Thisprocess is known in the art for some of the compounds encompassed byFormula XII, i.e., when the configuration at C-1 1 is alpha and theconfiguration at C-l5 is S. For the other compounds encompassed byFonnula XII, this reaction is novel, and novel Formula-XV and -XVIcompounds are produced.

For this carbonyl-to-hydroxy reduction, methods XIV XVI

the known ketonic carbonyl reducing agents which do not reduce ester oracid groups or carbon-carbon doubl e bonds. Examples of those are themetal borohydrides, especially sodium, potassium, lithium, and zincborohydrides, lithium (tri-tertbutoxy) aluminum hydride, metal trialkoxyborohydrides, e.g., sodium trimethoxyborohydride, and diisobutylaluminumhydride. The sodium, potassium, and zinc borohydrides are preferred forthis reduction, especially zinc borohydride.

Unexpectedly, the amalgamated aluminum metal found useful above intransforming the Formula-XI epoxides to Formula-XII hydroxy compoundshas also been found useful as an agent for this carbonyl-tohydroxyreduction of PGE-type compounds to PGF- type compounds. Either thePGE-type salts or the PGE- type esters are employed, for example theFormula-XII hydroxy compounds produced from the Formula-XI epoxides withor without intermediate isolation. Fur: thermore, the Formula-XIepoxides may be subjected to the combined epoxide-reduction andcarbonylreduction reactions practically simultaneously by operating athigher temperatures, for example 40-60 C., although it is preferred forhigh yields of the llhydroxy compounds that the reductions be donestepwise. .The solvents which are operable for this reduc- 29 tion aregenerally the same as those found useful for the epoxide-reduction step.Somewhat higher temperatures or longer reaction times are required forthe car bonyl-to-hydroxy reduction, however. For example, at 25 C.,about 4 to 24 hours are required; at higher temperatures, e.g., 5060 C.,about I to about 2 hours are sufficient.

This carbonyl reduction usually produces a mixture of PGF type and PGFtype compounds, i.e., compounds with the alpha configuration andcompounds with the beta configuration for the hydroxy at C-9. Thismixture of alpha and beta isomers is separated by methods known in theart, e.g., chromatography on silica gel. See Pike et al., ibid., forexample. If the Formula-XII starting material is a mixture of Ila andIIB isomers, then this reduction will usually produce four isomers,i.e., 9a,l la, 9a,l 15, 93,1 la, and 93,113. Those compounds are alsoseparated from such mixtures by silica gel chromatography.

Regarding the transformation of XII to XIV in Chart B, it will beobserved that the parameters for XII are such that all XII compounds areincluded in XIV. In other words, the transformation XII to XIV is anoptional process step in proceeding from XII to XV. The reason for thisis as follows. During the reduction of XIV to XV, the ratio of9a-hydroxy and 9B-hydroxy compounds formed will be different when R inXIV is hydrogen than when R is Si(A) For example, with the Formula-XIVcompound wherein R is hydrogen, G is and R O- represents HO i.e.,Ila-hydroxy, sodium borohydride reduction gives 42 parts of thecorresponding Formula-XV 9a-hydroxy compound, and 58 parts of the9B-hydroxy compound. But with the Formula-XIV compound wherein R ishydrogen, G is H o si cH,),,

and R-,O- represents (CH ,-SiO sodium borohydride reduction gives 85parts of the corresponding Formula-XV 9a-hydroxy compound and 15 partsof the 9,B-hydroxy compound. Similar difierences are observed with theother isomers encompassed by Formula XIV although not necessarily in thesame direction. Accordingly, whether R in Formula XIV is to be hydrogenor Si(A) depends on the particular formula XV C-9 isomer desired and theinfluence of silylation on the isomer ratio. For any particularFormula-XIV starting material, the latter is readily determined by smallscale reduction with and without silylatio'n: When silylation'beforecarbonyl reduction is indicated, largely for economic reasons, it ispreferred that A be methyl, i.e., that R be (Cl-I S i Thistransformation of XII to XIV to XIV wherein R is Si.-(A) is carriedi outas described above for the transformation of hydroxy to .OSi (A) at C-lprior to the Chart A reactions. When R is XII is hydrogen, the COOH isalso transformed in ,part or entirely to COOSi-(A) with prolongedsilylation and exces silylating agent. It is optional in transformingXII to XIV wherein R is Si(A) whether or not the 30 COOH of XII isesterified to COOSi--(A) When G in Formula XIV is H OH or the acetyl isremoved by alcoholysis also as described above for changing acetoxy atC-15 to hydroxy. These reactions are shown in Chart B as XV to XVI.

When R in Formula XVI is methyl and the compound wherein R is hyrogen isdesired, that methyl ester is saponified, by methods known in the art.See, for example, Just et al., J. Am. Chem. Soc. 91, 5371 (1969). Thissaponification also changes a C-l5 acetate to a C15 hydroxy.

The compounds encompassed by Formula XVI include the known compounds PGFPGF B and the methyl esters of those. Also included in Formula XVI arethe novel compounds l5B-PGF a ISBPGF B and the methyl esters of those.All of these new and old compounds are ll a-hydroxy compounds. Alsoincluded in Formula XVI are the corresponding but novel llB-hydroxycompounds which are also encompassed by Formula V and which are usefulfor the pharmacological purposes described above either as such ortransformed into salts or esters as described above.

When one of these Formula-XVI compounds has the R or epi configurationfor the hydroxy at C- l 5, and the corresponding compound with the Sconfiguration at C-l5 is desired, or when one of these Formula-XVIcompounds has the S configuration for the hydroxy at C-l5, and thecorresponding compound with the R or epi configuration at C-l5 isdesired, those desired compounds are made by the processes of Chart C.In Chart C, R R R B, and are as defined above.

The overall process scheme of Chart C is to start with one particularC-l5 isomer of a compound encompassed by Formula XVI, i.e., either 15(8)or 15(R). The C-l5 hydroxy of that isomer is oxidized to a ketoniccarbonyl (XVII). Then, after an optional silylation of the C-9 and C-1 1hydroxy groups (XVIII), the C-l5 carbonyl is reduced back to a secondaryhydroxy group. That reduction produces two C-l5 hydroxy isomers, onewith S configuration and one with R or epi configuration. After removalof any silyl groups, the isomers XIX and XX are separated. One of theisomers will be the same compound used as starting material (XVI).The-other isomer will be the desired product. The starting materialisomer is recycled to produce more of the desired isomer. This reactionscheme has previously been used to transform PGF, to 158- PGF a See Pikeet al., J. Org. Chem. 34, 3552 (1969).

CHART c (oxidation) (Si lylation) 1/ (reduction) i (hydrolysis) XVI XVII

XVIII XIX Referring now to Chart C, starting material XVI 60 vatedmanganese dioxide, or nickel peroxide (see (from Chart B) is a singlecompound, a mixture of two Fieser et al., Reagents for OrganicSyntheses, John compounds, one with alpha and one with beta configu-Wiley& Sons, lnc., New York, N.Y., 1967, pp. 215, ration at G9, or amixture of four compounds, i.e., 637, and 731). Alternatively, theseoxidations are car- 9a,l la, 9a,! 1,8, 9,8,1 1a, and 93,1113.

ried out by oxygenation in the presence of the 15- For the oxidation ofXVI to XVII, any oxidizing agent 65 hydroxyprostaglandin dehydrogenaseof swine lung can be used which will oxidize an allylic alcohol to an(see Arkiv for Kemi 25, 293 (1966)). These reagents a,B-unsaturatedketone or aldehyde. Examples of hose are used according to proceduresknown in the art. See, are 2,3-dichloro-5,6-dicyano-l,4-benzoquinone,actifor example, J. Biol. Chem. 239, 4097 (I964).

Regarding the transformation of XVII to XVIII in Chart C, it will beobserved that the parameters for XVIII are such that all XVII compoundsare included in XVIII. In other words, the transformation of XVII toXVIII is an optional process step in proceeding from XVII to XIX and XX.The reason for this is as follows. During the reduction of XVIII to XIXand XX, the ratio of XIX to XX obtained will be different when R, inXVIII is hydrogen than when R is Si(A) For example, reduction of theFormula-XVIII 901,1 la-isomer wherein R and R are both hyrogen and zincborohydride gives the corresponding Formula-XIX and -XX in the amounts43 parts of XIX (R or epi configuration) and 57 parts of XX (Sconfiguration). On the other hand, when R in the Formula-XVIII reactantis -Si- (A);,, the amounts with the same reducing agent are 27 parts ofXIX and 73 parts of XX. Similar differences are observed with the otherisomers encompassed by Formula XVIII although not necessarily in thesame direction. Accordingly, whether R in formula XVIII is to behydrogen or --Si-(A) depends on the particular C-l isomer desired andthe influence of silylation on the isomer ratio. For any particularFormula-XVII starting material, the latter is readily determined bysmall scale reductions with and without silylation. When silylationbefore carbonyl reduction is indicated, largely for economic reasons, itis preferred that A be methyl, i.e., that R, be (CI-I --Si.

These silylations are carried out as described above for the Chart A andChart B silylation.

The carbonyl reduction of XVIII to XIX is carried out as described abovefor the transformation of PGE- type Forrnula-XIV compounds to PGF-typeFormula- XV compounds. As for those reductions, the sodium, potassiumand zinc borohydrides are preferred as reducing agents, especially zincborohydride.

When the method used to isolate the carbonyl reduction product does notremove any Si(A) groups which may be present, that is accomplished asdescribed above for the removal of Si(A) groups from Formula-XIIproducts (Chart A, XII to XIII).

The Formula-XIX and -XX products are separated from each other bymethods known in the art, for example, silica gel chromatography. See,for example, Pike et al., J. Org Chem. 34, 3552 (1969) for this type ofseparation. v g

If one of the isomers or isomer mixtures of Formulas XIX or XX is notdesired for a pharmacological use: as such or transformed to esters orpharmacologically acceptable salts as described above, that isomer orisomer mixture is recycled as a Formula-XVI starting material in theprocesses of Chart C to produce additional of the desired isomer.

The products of Formulas XIX and XX wherein the configuration of the C-11 hydroxy is beta are encompassed by Formula V. The products of FormulaXIX wherein the configuration of the C-1 1 hydroxy is alpha areencompassed by Formula VI. The intermediates of Formula XVII areencompassed by Formula VII. Thus, all of the compounds are useful forthe pharmacological purposes described above for the Formula V, VI andVII compounds. The compounds prepared as in Chart C are also useful tomake the other esters and the pharmacologically acceptable salts of theFormula V, VI. and VII compounds also as described above.

There are two particular embodiments of the novel process of thisinvention which are especially preferred. One of those embodimentsprovides an optional route to PGF a and starts with ISB-PGA acetatemethyl ester, the most abundant component of Plexaura homomalla (Esper),1792, forma R. The other embodiment provides a preferred route of PGEand starts with PGA readily obtained as described above by maintainingcolonies or colony pieces of Plexaura homomalla (Esper), 1792, forma Sin contact with water in a temperature range up to 50 C. untilsubstantially free of PGA l5 acetate methyl ester.

CHART D ISB-P GA acetate methyl ester 1', (oxidation) l5,B-PGA acetatemethyl ester or and B 10,] l-epoxides l, (reduction) ISBPGE and l IB,l5fi-PGE IS-acetate methyl esters (separation) l5B PGE l5-acetatemethyl ester 15 B-PGFZ a a CHART E PGA (silylation) (oxidation) PGAlS-Si-(A); ether or and [3 10,1 l-epoxides (reduction, hydrolysis) P012;and l 1 ros,

(separation) PGE The first of these embodiments is shown in Chart D, andthe second is shown in Chart E. All of these Chart D and Chart Ereactions and reagents for effecting them are described generically andspecifically above, and all are exemplified below. In Charts D and E, itis preferred that Si-(A) be Si(CI-I Also in Charts D and E, it isoptional whether silylation of l5-oxo-PGF (Chart D) or PGA (Chart E)produces the corresponding Si-(A) esterether or only 35 the ether.

The invention is more fully understood by the following Examples andPreparations:

All temperatures are in degrees Centigrade.

Ultraviolet spectra are recorded on a Cary Model 15 spectrophotometer.

The collection of chromatographic eluate fractions starts with theeluant front reaches the bottom of the column. Brine, herein, refers toan aqueous saturated sodium chloride solution.

The A-lX solvent system used in thin layer chromatography (TLC) is madeup from ethyl acetate-acetic acid-2,2,4-trimethylpentane-water(90:20:50z100) according to M. Hamberg and B. Samuelsson, J. Biol. Chem.241, 257 (1966).

Preparation 1 To distinguish Plexaura homomalla (Esper), 1792, forma Rfrom Plexaura homomalla (Esper), 1792, forma S, a TLC method is used. Aspecimen approxi mately 2 cm. in length is harvested and placed in asmall vial, with a small amount of water if necessary to insure it iswet, and kept closed for 6-24 hrs. About one ml. of methanol is thenadded and the sample is either shaken for 2 hrs. at about 25 C. or isstored for 16-24 hrs. at about 10 C. A sample of the liquid (IO-21 A) isspotted on a TLC plate. It is preferred to use a fluorescent-treatedsilica gel plate, e.g. Uniplate Silica Gel GF (Analtech, Inc., Newark,Del.). As reference standards, spots of PGA and B-PGA are also applied.The plate is developed in the A-lX system (Hamberg and Samuelsson, J.Biol. Chem. 241, 257 (1965)). The spots are finally visualized withvanillin-phosphoric acid spray (McAleer, Arch. Biochem. E. Biophys. 66,120 1957)). Comparison of the unknown with the two reference spots isthen made and the identity of the coral established (forma Scorresponding to PGA forma R to ISB-PGA Preparation 2 PGA from Plexaurahomomalla (Esper), 1792 forma S Colonies of Plexaura homomalla (Esper),1792, forma S, collected from reefs off the north shore of Jamaica, arefrozen by contact with solid carbon dioxide within 1 hour after removalfrom the reef waters. The frozen colonies are maintained in insulatedboxes containing solid carbon dioxide (temperature below about C.) untilready for thawing. Then, the frozen colonies (700g.) are ground to asmall particle size (Waring blender) and mixed with 1500 ml. of water.The mixture is maintained about 20 hrs. at about C. with stirring. Then,the mixture is filtered through a pad of diatomaceous earth, and thefiltrate is acidified with concentrated hydrochloric acid to pH about2-3. The acidified filtrate is extracted four times with ethyl acetate.The extracts are combined, filtered, washed with brine, dried withanhydrous sodium sulfate, and evaporated under reduced pressure to give11 g. of oily residue.

The solid residue on the diatomaceous earth filter pad is stirred 2hours in methanol (enough to cover said residue) at 25 C. The mixture isthen filtered, and the filtrate is evaporated to give 14 g. of oilyresidue.

The two oily residues are combined and chromatographed on 1500 g. ofacid-washed silica gel, eluting successively with 8 l. of a 25 to 65%gradient of ethyl acetate in Skellysolve B, 8 l. of a 65 to 100 gradientof ethyl acetate in Skellysolve B, and 5 l. of 2% methanol in ethylacetate, collecting 500 ml. fractions. (Skellysolve B is a mixture ofisomeric hexanes). Fractions 8-12 are combined and evaporated to give asmall amount of PGA containing a trace of PGA methyl ester. Fractions15-18 are combined and evaporated to give 9.54 g. of PGA Fractions 3540are combined to give 0.414 g. of PGE Preparation 3 l5B-PGA from PlexauraHomomalla (Esper), 1792, forma R Colonies of Plexaura homomalla (Esper),1792, forma R, collected from reefs off the southeast shore of Floridanear Miami, are chopped into chunks several inches long. The chunks arefrozen by contact with solid carbon dioxide with one hour after removalfrom the reef waters. The frozen colony pieces are maintained ininsulated boxes containing solid carbon dioxide (temperature below about20 C.) until ready for thawing. Then, colony pieces (600 g.) are mixedwith 1500 ml. of water. The mixture is stirred and maintained at 25 C.for 23 hours. The mixture is then filtered through a pad of diatomaceousearth, and the filtrate is acidified to pH about 2-3 with concentratedhyrochloric acid. The acidified filtrate is extracted four times withethyl acetate. The extracts are combined, filterd, washed with brine,dried with anhydrous sodium sultate, and evaporated to give 9.2 g. ofoily residue.

The solid residue on the diatomaceous earth pad is stirred 15 hours inmethanol (enough to cover said residue) at 25 C. The mixture is thenfiltered, and the filtrate is evaporated. The residue is dissolved inethyl acetate, and the solution washed successively with 3 Nhydrochloric acid and brine, dried with anhydrous sodium sulfate, andevaporated to give 5.83 g. of an oily residue.

The second oily residue and 8.2 g. of the first oily residue arecombined and chromatographed on one kg. of acid-washed silica gel,eluting successively with 3-1. portions of 25%, 35%, 45%, 55%, and 65%ethyl acetate in Skellysolve B, collecting SOO-ml. fractions. Fractions18-22 are combined and evaporated to give 5.54 g. of l5B-PGA Fractions15-17 are combined and evaporated to give 1.37 g. of lSB-PGA methylester.

Preparation 4 PGA compounds from Plexaura homomalla (Esper), 1792, formaS Frozen colonies of Plexaura homomalla (Esper), 1792, forma S (seePreparation 2) are broken manually into pieces several cm. in length.The pieces (500 g.) are then covered with methanol and the mixture ismaintained for 3 hours at 25 C. The mixture is then ground in a Waringblender and filtered, and the filtrate is evaporated under reducedpressure. The residue is dissolved in ethyl acetate, and the solution iswashed successively with one N hydrochloric acid, water, and brine,dried with anhydrous sodium sulfate, and evaporated under reducedpressure. The oily residue is chromatographed on 2 kg. of acid-washedsilica gel wetpacked with Skellysolve B (a mixture of isomeric hexanes),eluting with 24 l. of a 25 to ethyl acetate in Skellysolve B gradient.The fractions which contain PGA acetate methyl ester, PGA acetate, PGAmethyl ester, and PGA as shown by TLC with the A-lX system areseparately combined and evaporated to give those compounds.

Preparation l5B-PGA compounds from Plexaura homomalla (Esper), 1792,forrna R Colonies of Plexaura homomalla (Esper), 1792, forma R,collected from reefs off the southeast shore of Florida near Miami, arechopped into chunks several inches long. The chunks are frozen bycontact with solid carbon dioxide within one hour after removal from thereef waters. The frozen colony pieces are maintained in insulated boxescontaining solid carbon dioxide (temperature below about C.) until thetime for extraction. Then, the frozen colony pieces are ground to asmall particle size (Mitts and Merrill hogger; average largest dimensionabout 5 mm). The particles (1500 g.) are then stirred at high speed with5 gallons of dichloromethane for 20 minutes at about C. externaltemperature. The mixture of dichloromethane and particles is thenfiltered through a pad of diatomaceous earth, and the filtrate isevaporated to about a 2-liter volume at C. under reduced pressure. Theliquid which remains is washed with water, dried with sodium sulfate,and evaporated at 30 C. 'under reduced pressure.

The oily residue (60 g.) is chromatographed on 3 kg. of silica gel wetpacked in Skellysolve B (a mixture of isomeric hexanes), elutingsuccessively with a gradient of 4 1. of Skellysolve B and 4 l. of 20%ethyl acetate in Skellysolve B, 27 l. of 20%, 18 l. of 50%, and 8 l. of75% ethyl acetate in Skellysolve B, collecting 600-ml. fractions.Fractions 39-60 are combined and evaporated to give 24.3 g. of 15B-PGAacetate methyl ester. Between fractions 60 and 74 those fractions shownby TLC to contain 15B-PGA acetate are combined and evaporated to yieldthat compound. Fractions 74-76 are combined and evaporated to give 1.03g. of 153- PGA methyl ester. Fractions 83-91 are combined and evaporatedto give 1.08 g. of l5B-PGE 15-acetate methyl ester. Still laterfractions shown by TLC to contain 15/3-PGE methyl ester are combined andevaporated to yield that compound.

Detection of the respective compounds by TLC is done by methods known inthe art, e.g. by spotting the extract fractions on a TLCsilica gel platealongside spots of the authentic compounds, developing the plate withthe A-lX system, and observing which spots of the extract fractionscorrespond exactly to the spots of the authentic compounds.

Following the procedures of Prepartion 5, but substituting Plexaurahomomalla (Esper), 1792, forrna S for the Plexaura homomalla (Esper),1792, forrna R of that example, there are obtained the correspondingcompounds of 15(S) configuration, viz.: PGA acetate methyl ester, PGAacetate, PGA methyl ester, PGE l5-acetate methyl ester, and PGE methylester.

Preparation 6 PGA and 5,6-trans-PGA Separation of PGA from 5,6-trans-PGAis done on a chromatographic column using a silver-saturatedion-exchange resin. Preferably a macroreticular ion exchange resin isused, e.g. a sulfonated styrene-divinylbenzene copolymer having surfacearea of 40-50 sq. m./g., 30-40% porosity, and total exchange capacity of4.5-5.0 meq. per gram of dry resin, for example Amberlyst 15, availablefrom Rohm and Haas Co., Philadelphia, Pa. The acid-form resin is packedin a column,

washed with warm water, and converted to the silver form by passing a10% silver nitrate solution through the column until the effluent showsa pH of 3.5-4.0. The column is then washed with water to remove ionicsilver, and finally with denatured ethanol (Type 3A). A solution of amixture of PGA and 5,6-trans-PGA e.g. fractions 15-18 of Preparation 2,in ethanol is charged to the column. Elution with BA alcohol then yieldsfractions which are combined according to their content of 5,6trans-PGA(faster-eluting) or PGA Testing for the presence of 5,6-trans-PGA or PGAin the eluate is conveniently done by TLC using silver nitratetreatedsilica gel plates (e.g. Analtech Uniplates dipped in saturated ethanolicsilver nitrate and dried) and developing with the A-IX system. R, of PGAis 0.45; R, of 5,6-trans-PGA 2 is 0.50. Combined fractions areconcentrated, partitioned between dichloromethane and a little water,dried over sodium sulfate, and concentrated under reduced pressure toyield the title compounds.

For quantitatively assaying the 5,6-trans-PGA content of mixtures of PGAand 5,6-trans-PGA a combination thinlayer-spectrophotometric assay isused. Silica gel-impregnated glass microfiber sheets (e.g. lTLC sheetsof the Gelman Instrument Co., Ann Arbor, Mich. are impregnated with asilver nitrate, using 5% ethanolic silver nitrate and drying. Spots of100 to 200 ,ug of the PGA mixture are applied and developed in thesolvent system 2,2,4-trimethylpentane:ethyl acetate: acetic acid: water(100:35z8210, upper phase).

- The sheet is dried and sprayed with Rhodamine 6G (Applied Science Co.,State College, Pa) and viewed under ultraviolet light. The areascontaining the cis and trans materials (R; of PGA 0.6; R; of 5,6-trans-PGA 0.7) are marked, then excised and eluted with methanol (1.9 ml.) andpotassium hydroxide solution (0.1 ml. of 45%). After incubation at 40for 30 min., the respective solutions are centrifuged and analyzedspectrophotometrically at 278 nm.

Following the procedure of Preparation 15B-PGA is separated from l5B-PGAPreparation 7 6, 5,6-trans- Preparation 8 PGA l5-Acetate Methyl Ester,separation from 5,6- Trans-PGA 15-Acetate Methyl Ester.

A mixture of PGA 15-acetate methyl ester and 5,6- trans-PGAIS-acetatemethyl ester (11.0 g., :15 is dissolved in 415 ml. of asolution of methanol-wateracetic acid (-5-04) and mercuric acetate (6.1g.), and left standing at about 25 C. for 30 min. Water (250 ml.) isadded and the mixture extracted twice with 700 ml. of Skellysolve B. TheSkellysolve B phase is washed with ml. of 60% methanol, dried oversodium sulfate, and concentrated to an oil (4.35 g.) hav-

1. A COMPOUND OF THE FORMULA
 2. A compound according to claim 1 whereinthe hydroxy is attached to the ring in alpha configuration.
 3. Acompound according to claim 1 wherein the hydroxy is attached to thering in beta configuration.